Receptor Tyrosine Kinases (RTK) and G-protein coupled receptors (GPCR) are the two most widely studied cell signaling hubs in eukaryotes. For several decades these two pathways were believed to operate in a discrete mode by transducing signals through their respective downstream intermediates; upon ligand stimulation RTKs propagate the signals to the interior of the cell via adaptor proteins that are recruited to phosphotyrosines on the receptor tail (1), whereas GPCRs, which are 7-transmembrane (TM) receptors with an intrinsic Guanine nucleotide Exchange Factor (GEF) activity recruit and activate G proteins by triggering the exchange of GDP with GTP nucleotide (2). Gathering evidence over time has unraveled a complex cross-talk between these two pathways at multiple tiers (3, 4). For example, transactivation of RTKs by GPCRs via scaffolding proteins such as β-arrestins (5) is a well-documented and widely-accepted phenomenon. Numerous studies have also provided evidence to support the reverse concept, i.e., transactivation of heterotrimeric G proteins by growth factors (6). However, it was not until recently that this concept gained traction with the discovery and characterization of Gα-Interacting Vesicle associated protein (GIV; a.k.a Girdin), an unusual signal transducer that can bind both RTKs and G proteins.
GIV is a multi-modular (FIG. 1A) signal transducer and a GEF for Gαi (7). Working downstream of a variety of growth factors [EGF (8, 9), IGF (10), VEGF (11), Insulin (7, 12, 13) and PDGFR (14)] GIV modulates, i.e., either enhances, or suppresses a variety of signaling pathways, all via its ability to activate Gαi in the close proximity of a ligand-activated RTK (7). Multiple studies (summarized in FIG. 5 (15)) employing a selective GEF-deficient GIV mutant (F1685A) have demonstrated that the signaling network downstream of RTKs in cells with wild-type GIV is a mirror image of the network in cells expressing a GEF-deficient mutant GIV. It is because cells can alter (increase or decrease) the levels of GIV mRNA/protein or selectively modulate GIV's GEF activity to modulate growth factor signaling pathways across a range of intensities (16), GIV is considered as a cellular “rheostat” for signal transduction (17). Consistent with its ability to integrate signals downstream of multiple receptors, GIV modulates growth factor signaling during diverse biological processes (17), e.g., cell migration, chemotaxis (13), invasion (18), development (19), self-renewal (20), apoptosis (14, 21) and autophagy (12). Increasing evidence also supports the clinical significance of GIV-dependent signaling during diverse disease processes (17); e.g., pathologic angiogenesis (11), liver fibrosis (14), nephrotic syndrome (21), vascular repair (22) and tumor metastasis (23).
The molecular mechanisms that govern how GIV influences a diverse range of pathophysiologic processes and how it may couple activation of G protein to multiple receptors have come to light only recently, at least in the context of a numerous RTKs that signal via GIV. GIV-dependent growth factor signaling appears to rely heavily on the unique multi-modular nature of its C-terminus (CT), within which two unlikely domains coexist—1) a previously defined GEF motif via which GIV binds and activates Gi (7) and 2) a newly defined ˜110 a stretch which folds into a SH2-like domain in the presence of phosphotyrosine ligands; the latter is necessary and sufficient to recognize and bind specific sites of autophosphorylation on the receptor tail (9, 24). Thus, GIV serves as a platform that links RTKs to G proteins within RTK-GIV-Gαi ternary complexes only when both its GEF and SH2-like modules are intact. In the absence of either of these modules, ligand-activated RTKs and Gαi are uncoupled, and the recruitment of Gαi to RTKs and subsequent activation of G proteins is impaired.
Most common diseases, e.g., cancer, inflammation, diabetes are driven by multiple cell surface receptors that trigger and sustain a pathologic signaling network. The largest fraction of therapeutic agents that target individual receptors/pathways often eventually fails due to the emergence of compensatory mechanisms. In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G-protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multi-modular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). It was recently demonstrated that GIV's C-terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for non-canonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach.
The discovery of coexisting SH2-like and GEF modules in-tandem within GIV-CT supported the idea that GIV's C-terminus has the necessary modular make-up to serve as a platform for convergent signaling downstream of multiple RTKs via G proteins. However, it was not possible to visualize this platform until recently, when genetically encoded fluorescent biosensors comprised of these two modules within GIV-CT were developed. These biosensors revealed that the evolutionarily conserved C-terminus of GIV represents the smallest, functionally autonomous unit that retains most key properties of full length GIV (25), i.e., 1) they can bind and activate Gαi in cells in a GEF dependent manner; 2) they retain the properties of receptor recruitment and signal transduction characteristic of full length GIV; 3) they serve as a bona fide platform for assembly of RTK-Gαi complexes at the PM and for non-canonical activation of Gαi in response to growth factors; and 4) they are sufficient to trigger cell migration/invasion through basement membrane matrix. Thus, comprised of the essential modules (GEF and SH2-like domains), GIV-CT is sufficient for linking G proteins to RTKs, for triggering G protein activation in the vicinity of ligand-activated RTKs, for modulation of growth factor signaling, and for triggering complex cellular processes like cell invasion.
Despite the emergence of GIV-CT as the long-sought platform for non-canonical transactivation of G proteins by multiple growth factor RTKs, exogenous manipulation of this platform has remained out of reach. There is currently no existing art of disrupting GIV-Gi axis of signaling exogenously; no current knowledge of how such disruption may affect signaling and cell behavior; and/or no current method of modulating multi-receptor driven pathologic signaling.
Insulin resistance (IR) is a metabolic disorder in which adipocytes and muscle cells fail to take up and metabolize glucose in response to the hormone insulin. Although IR is a hallmark of Type II Diabetes Mellitus (T2DM), IR alone in the absence of T2DM significantly increases the risk for stroke, heart failure and atherosclerosis (Carter, 2005; Rundek et al, 2010).
Although multiple etiologic factors contribute to the pathogenesis of IR (Saltiel & Kahn, 2001), they all ultimately converge to suppress critical components of metabolic insulin signaling. Insulin binds its receptors (InsR, IGF1R), which triggers receptor autophosphorylation, and subsequent tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), amongst others. This leads to the recruitment and activation of Src-Homology-2 (SH2) proteins such as p85α(PI3K) and downstream activation of Akt (Taniguchi et al, 2006). Akt triggers the translocation of the 12-transmembrane glucose transporter 4 (GLUT4) to the plasma membrane (PM) by phosphoinhibiting the Rab GTPase activating protein (GAP) AS160 (Miinea et al, 2005). Among the many adaptors that relay signals within the insulin cascade, IRS1 is widely believed to serve as the major node for orchestrating metabolic insulin signaling (Taniguchi et al, 2006).
Besides IRS1, metabolic insulin signaling relies also on the activation of heterotrimeric G proteins, another major hub in eukaryotic signal transduction. InsRs are functionally coupled to the pertussis-toxin sensitive Gαi/o proteins, e.g., insulin can trigger their activation (Ciaraldi & Maisel, 1989; Rothenberg & Kahn, 1988), localization (Gohla et al, 2007) and phosphorylation (Krupinski et al, 1988; O'Brien et al, 1987). Activation of Gi augments insulin sensitivity (Chen et al, 1997; Song et al, 2001), enhances tyrosine phosphorylation of both InsR and IRS1 (Moxham & Malbon, 1996) and triggers efficient translocation of GLUT4 storage vesicles (GSVs) to the PM (Ciaraldi & Maisel, 1989; Kanoh et al, 2000; Song et al, 2001). Although numerous clues consistently point to a critical role of Gi activation in the insulin response, who/what couples and activates Gi downstream of InsR, and how such activation may cross-talk with IRS1-dependent insulin signaling and trigger downstream metabolic events remain unknown. Additionally, little is known about how G protein pathways are altered in IR.
With regard to the pathogenesis of IR, suppression of metabolic insulin signaling via the IRS1/PI3K pathway is an invariable hallmark (Kahn & Flier, 2000; Le Roith & Zick, 2001; Pessin & Saltiel, 2000). Such suppression occurs via common mechanisms that involve cellular accumulation of lipid metabolites (acyl-CoAs, ceramides, and diacyglycerol, etc), which activate, among many other kinases, the critical protein kinase C-Theta (PKCθ) (Griffin et al, 1999; Yu et al, 2002). PKCO dependent phosphoinhibition of IRS1 at Ser1101 (Li et al, 2004) is considered an important event that triggers lipid-induced IR. PKCθ expression levels are increased in the skeletal muscles of obese diabetics and hold an inverse relationship to insulin sensitivity (Schmitz-Peiffer et al, 1997; Yu et al, 2002), and PKCθ−/− null mice demonstrate a protective effect against IR despite a high fat diet (Kim et al, 2004). These studies and many others have shaped the paradigm that IR is triggered when IRS1 is phosphoinhibited by kinases like PKCθ. However, some recent studies have revealed inconsistencies in this paradigm [summarized in (Hoehn et al, 2008)]. Emerging evidence indicates that IRS1 is insufficient for orchestrating the insulin response (Krook et al, 1996), and that multiple RTKs can trigger IR independent of IRS1 (Hoehn et al, 2008). These studies raise the possibility that major unidentified signaling nodes exist within the insulin signaling cascade, whose inhibition via the fatty-acid/PKCθ pathway triggers IR.
GIV is a Guanine-nucleotide Exchange Factor (GEF) which activates Gαi1/2/3 (Garcia-Marcos et al, 2009), contains a SH2-like domain that directly binds InsR (Lin et al, 2014), is a direct substrate of InsR which phosphorylates GIV at Y1764 (Lin et al, 2011), is a bona-fide enhancer of the PI3K-Akt pathway downstream of InsR and other RTKs (Lin et al, 2011) and is a substrate for PKCθ; the latter phosphorylates and inhibits signaling via the GIV-Gαi axis (Lopez-Sanchez et al, 2013). Furthermore, a recent study has indicated that GIV may serve as a major regulator of the metabolic insulin response in skeletal muscles (Hartung et al, 2013); overexpression of GIV in myoblasts leads to hyperphosphorylation of IRS1 and enhanced glucose uptake, whereas depletion of GIV suppresses both. Despite these insights, the molecular mechanisms that enable GIV to enhance the metabolic insulin-IRS1 response in physiology or mechanisms that derail this pathway in the setting of IR remained unknown.